Phthalazino[1,2-b]quinazolinones as p53 Activators: Cell Cycle Arrest, Apoptotic Response and Bak-Bcl-xl Complex Reorganization in Bladder Cancer Cells

Article abstract of DOI:10.1021/acs.jmedchem.6b01769

p53 inactivation is a clinically defined characteristic for cancer treatment-nonresponsiveness. It is therefore highly desirable to develop anticancer agents by restoring p53 function.1 Herein the synthesized phthalazino[1,2-b]quinazolinones were discovered as p53 activators in bladder cancer cells. 10-Bromo-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one (5da) was identified as the most promising candidate in view of both its anticancer activity and mechanisms of action. 5da exhibited strong anticancer activity on a broad range of cancer cell lines and significantly reduced tumor growth in xenograft models at doses as low as 6 mg/kg. Furthermore, 5da caused cell cycle arrest at S/G2 phase, induced apoptosis, changed cell size, and led to cell death by increasing the proportion of sub-G1 cells. Molecular mechanism studies suggested that accumulation of phospho-p53 in mitochondria after 5da treatment resulted in conformational activation of Bak, thereby evoking cell apoptosis, finally leading to irreversible cancer cell inhibition. Our present studies furnish new insights into the molecular interactions and anticancer mechanisms of phospho-p53-dependent quinazolinone compound.

Full text of DOI:10.1021/acs.jmedchem.6b01769

Subscriber access provided by UNIV OF NEWCASTLE
Article
Phthalazino[1,2-b]quinazolinones as p53 Activators:
Cell Cycle Arrest, Apoptotic Response and Bak–Bcl-
xl Complex Reorganization in Bladder Cancer Cells
Guo-Hai Zhang, Jing-Mei Yuan, Gang Qian, Chen-Xi GU, Kai Wei, Dong-Liang
Mo, Jiang-Ke Qin, Yan Peng, Zuping Zhou, Cheng-Xue Pan, and Gui-Fa Su
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01769 • Publication Date (Web): 26 Jul 2017
Downloaded from http://pubs.acs.org on July 26, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Phthalazino[1,2-b]quinazolinones as p53 Activators: Cell Cycle Arrest,
Apoptotic Response and Bak–Bcl-xl Complex Reorganization in Bladder
Cancer Cells
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Guo-Hai Zhang†,‡,#, Jing-Mei Yuan†,#, Gang Qian†, Chen-Xi Gu†, Kai Wei†,
Dong-Liang Mo†, Jiang-Ke Qin†, Yan Peng†, Zu-Ping Zhou‡, Cheng-Xue Pan*,†,
Gui-Fa Su*,†
†State Key Laboratory for Chemistry and Molecular Engineering of Medicinal
Resources, Ministry of Science and Technology of China; School of Chemistry and
Pharmaceutical Sciences, Guangxi Normal University, 15 Yu Cai Road, Guilin
541004, China.
‡Guangxi Universities Key Laboratory of Stem Cell and Biopharmaceutical
Technology, School of Life Sciences, Guangxi Normal University, 15 Yu Cai Road,
Guilin 541004, China.
TITLE RUNNING HEAD: Quinazolinones as p53 activators
KEYWORDS: quinazolinones, p53, conformational activation of Bak, apoptosis
*Corresponding Authors:
Professors Cheng-Xue Pan and Gui-Fa Su
Tel. & Fax: +86 773 5826869, Email: chengxuepan@163.com (C.-X. Pan);
gfysglgx@163.com (G.-F. Su)
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 56
1
2
3
4
5
6
7
8
9
ABSTRACT: p53 inactivation is a clinically defined characteristic for cancer
treatment-nonresponsiveness. It is therefore highly desirable to develop anticancer
agents by restoring p53 function.1 Herein the synthesized phthalazino[1,2-b]-
-quinazolinones were discovered as p53 activators in bladder cancer cells.
10-Bromo-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one (5da)
was identified as the most promising candidate in view of both its anticancer activity
and mechanisms of action. 5da exhibited strong anticancer activity on a broad range
of cancer cell lines and significantly reduced tumor growth in xenograft models at
doses as low as 6 mg/kg. Furthermore, 5da caused cell cycle arrest at S/G2 phase,
induced apoptosis, changed cell size and led to cell death by increasing the proportion
of sub-G1 cells. Molecular mechanism studies suggested that accumulation of
phospho-p53 in mitochondria after 5da treatment resulted in conformational
activation of Bak, thereby evoking cell apoptosis, finally leading to irreversible cancer
cell inhibition. Our present studies furnish new insights into the molecular interactions
and anticancer mechanisms of phospho-p53-dependent quinazolinone compound.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
INTRODUCTION
6
7
8
9
Bladder cancer (BC) continues to be the second most common malignancy of
urinary system worldwide with an overall 5-years recurrence rate up to 78%.2-4
Especially for patients suffering from muscle-invasive tumors, high mortality is a
serious health threat due to distant metastases.5,6 Chemotherapy is commonly adopted
to delay relapse and prolong survival.7 However, no more than half of the
muscle-invasive BC patients respond to chemotherapy.8,9 Furthermore, the problem of
easy recurrence remains to be unsolved.10 Therefore, it is very important to explore
novel chemotherapeutic agents with treatment and prevention functions and elucidate
the mechanisms of increasing treatment responsiveness of bladder cancer cells.
Inactivation of the p53 tumor suppressor pathway is a common feature of human
cancers, furnishing an attractive avenue for cancer therapies based on restoring p53
function in established tumors.11,12 It has been proposed that p53 activation promoted
cancer cell killing.13,14 Moreover, activation of p53 pathway can restrict malignant
transformation by triggering apoptosis or cell cycle arrest.15 It is therefore highly
desirable to restore p53 function by persistent p53 activation with small-molecule
compounds for BC therapy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Quinazolinone is a key structural motif in many natural products, as well as in
synthetic pharmaceutical compounds. Quinazolinone derivatives exhibit diverse
pharmacological activities and have been identified as enzyme inhibitors,16-18
antileishmanial agents,19 receptor antagonists,20,21 anti-inflammatory agents22,23 and
anticancer agents.24-26 A recent study27 aimed to identify p53 activator with potent
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 56
1
2
3
4
5
6
7
8
9
antitumor activity documented that 5-(3-dimethylamino-propylamino)phthalazino-
-[1,2-b]quinazolin-8-one (BMH-727, 5ab, Figure 1) could regulate the p53 signaling
pathway and exhibit anticancer activities in vitro and in vivo. However, the synthesis
and further structure activity relationship studies of this scaffold have not yet been
reported in the literature. In order to obtain increased efficacy, in the present study, we
designed and synthesized a series of phthalazino[1,2-b]quinazolinone derivatives (5)
and identified 10-bromo-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazo-
-lin-8-one (5da, Figure 1) as the most promising one in view of both the anticancer
activity and the mechanisms of action. We were able to demonstrate clearly that 5da
could induce phospho-p53 mitochondria-targeted accumulation with complicated
regulatory process in cell apoptosis and proliferation inhibition.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1
RESULTS
Chemistry. Synthesis of phthalazino[1,2-b]quinazolinone derivatives 5 was
described in Scheme 1 and Scheme 2. First, commercially available anthranilic acids
were converted to the corresponding anthranilic hydrazides (2) in two steps according
to literature methods.28 Then 2 and phthalic anhydride in glycol were heated in oil
bath to provide 10-substituted 6H-phthalazino[1,2-b]quinazolin-5,8-diones (3) in high
yield. Treatment of 3 with phosphorus oxychloride gave 10-substituted 5-chloro-
-phthalazino[1,2-b]quinazolin-8-ones (4) in 50-72% yields. Finally, 4 reacted with
4
ACS Paragon Plus Environment

Page 5 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
amine derivatives in toluene furnished the target compounds 5. The structure of
10-fluoro-5-(2-dimethylaminoethylamino)phthalazino[1,2-b]quinazolin-8-one (5ba)
was further unambiguously determined by the X-ray crystallography (detailed
parameters are included in the Supporting Information).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1
Scheme 2
In Vitro Biological Evaluations. Compounds 5 were evaluated in vitro against a
panel of five cancer cell lines including T24, HepG2, NCI-H460, MGC-803 and HeLa
using microculture tetrazolium (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-
-zolium bromide) assay. The IC50 values derived from dose−response curves were
summarized in Table 1.
Table 1
As shown in Table 1, many of the compounds showed potent cytotoxicity to all
the tested cell lines. The data indicated that the substituent on the 5-position of the
phthalazino[1,2-b]quinazolin-8-one skeleton had a very important effect on activity.
Thirteen different substituents on the 5-position were explored. The results indicated
that in general aminoalkylamine side chains could give better antiproliferative activity
than 4-methyl-N-piperazinyl (series 5xe). And if the 5-substituent was replaced by a
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 56
1
2
3
4
5
6
7
N-morpholinyl (series 5xd), alkyl (5dk) or ω-alkoxycarbonyl substituted alkyl (5dl
and 5dm), the compounds almost lost the cytotoxicity.
8
9
The tail of the aminoalkylamine side chain also significantly affected the
antiproliferative activity. Compound with dimethylamino at the tail (5da) are more
potent than with diethylamino (5df) or diiospropylamino (5dg). A tail with
pyrrolidinyl and pyridyl (compounds 5dh and 5di) also result in potent
antiproliferative activity. When the tail of the aminoalkylamine was replaced by a
N-morpholinyl (5dc, 5dj), the cytotoxicity of the compounds would decrease. The
length of the side chain also showed notable influence to cytotoxicity. A link of
aminoethyl is more favorable than an aminopropyl (5da vs. 5db, 5dj vs. 5dc).
The data in Table 1 also suggested that replaced the hydrogen (H) atom (5ax
series) at the 10-position of the phthalazino[1,2-b]quinazolin-8-one skeleton with a
halogen atom (5bx-5dx series) or a methyl (5ex series), could increase the
cytotoxicity. Among them the Br substitution (5dx series) gave the most potency to
most of the tested cell lines in general, as exemplified by 5da. The anticancer
activities of 5da in vitro were superior to the first-line chemotherapeutic agent
10-hydroxycamptothecin (HCPT) and this compound was chosen to further
investigate the mechanism of its cytotoxic effect.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5da is not Pan Assay Interference Compounds (PAINS). Prior to study the
anticancer mechanism of 5da, firm experiments had been carried out to check its
apparent activity was not an artifact. 5da and its analogues are known as quinoline
class of putative PAINS, their absorption of light in the range 570-620 nm would
6
ACS Paragon Plus Environment

Page 7 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
interfere the effective IC50 values in turn.29 To check the potential interference effect,
we tested the absorption change of light in 490 nm in T24 cells after 5da treatment.
As shown in Figure 1B, 5da treatment (doses from 1 to 10 μM, 1 h) did not affect the
absorption of light in 490 nm (P>0.05). Another mechanism of assay interference of
such class putative PAINS may be to cause DNA damage. In order to test the
possibility, DNA relaxation assay was used to investigate the interaction between 5da
and DNA directly. After concentrations (1-10 M) of 5da treatment, there was nearly
no difference either in conformational changes of DNA or in the mobility rate of DNA
compared with the negative control group; while in the positive control group (EB
group), the migration rate of DNA was significantly inhibited (Figure 1C). Moreover,
a biomarker, γH2AX was adopted to detect DNA damage. As depicted in Figure 1D,
no DNA damage was detected after 5da treatment. Furthermore, 5da containing a
fluorophore could emit fluorescence in the range 420-450 nm (Figure 1E), thus
leading to other mechanisms of assay interference. To test the possibility, T24 cells
were treated with 5da (doses from 1 to 10 μM, 1 h), stained with PI and examined by
FACS analysis. As shown in Figure 1F, 5da neither triggered obvious membrane
disruption nor affected the distribution of cells in the FITC channel to further ensure
the reliability of the data based on fluorescence analysis in the following experiments.
Collectively, these results suggested that 5da is not PAINS.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5da Induces Cell Cycle Arrest and Cell Size Change in T24 cancer cells. To
study the potential mechanistic pathways responsible for cell proliferation inhibition
by 5da, we tested the change of cell cycle in T24 and J82 cancer cell lines. As shown
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 56
1
2
3
4
5
6
7
8
9
in Figure 2A and D, 5da treatment (doses from 1 to 10 μM, 48 h) clearly increased the
cell proportion of S/G2 phase and sub-G1 phase (a specific phase of cell death), while
the cell proportions in G1 phase were markedly reduced (Figure 2B and D).
Consistent with the effects on cell cycle arrest, 5da induced a more profound
reduction in cell size (Figure 2C, left). The relative cell size (mean FSC-H) of
5da–treated cells were 87.7% of control cells in T24 cell line (Figure 2C, right).
Collectively, these results documented the antiproliferation potency of 5da in a broad
spectrum of cancer cell lines were potentially due to the S/G2 phase arrest-induced
growth inhibition and cell size reduction.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2
To investigate the mechanism of 5da-induced cell cycle arrest, we firstly checked
the p53 expression and p53 phosphorylation after 5da treatment, as the 5da analogue
5ab could pharmacologically activate the p53 pathway to exhibit anticancer activity.27
As shown in Figure 2E, both 5da and 5ab could induce p53 activation by promoting
p53 phosphorylation in a dose-dependent manner along with little impact on p53
expression. To find out whether the anticancer effect of 5da against T24 cancer cells
was due to p53 activation, we examined the anticancer activities of 5da in DU145
cancer cells (p53 mutation cell line), p53-null PC-3 cancer cells and LnCap cancer
cells (wild-type p53). Our results documented that the anticancer activities of 5da in
DU145 cancer cells and PC-3 cancer cells were obviously attenuated compared to that
8
ACS Paragon Plus Environment

Page 9 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
in wt-p53 LnCap cancer cells (Figure 2F). To further explore that the inhibitory effect
of 5da on cell proliferation was mainly due to p53 activation, p53-siRNA was used to
ablate endogenous p53. Both cells (p53 present and absent) were exposed to different
concentrations of 5da for 48 h. In scRNA-treated T24 cells and control group, 5da
treatment dramatically inhibited cell proliferation in a dose-dependent manner, while
in p53-ablated T24 cells, the sensitivity of T24 cells to 5da was significantly
decreased (Figure 2G). Furthermore, to provide more direct evidence on p53
activation, we also studied the inhibition of interactions between p53 and MDM2 after
5da treatment. As depicted in Figure 2H, both 5da and nutlin-3 effectively activated
p53 in T24 cells, evident by robust increase of p21 protein. 5da was even more potent
than nutlin-3, the first MDM2 inhibitor reported by Vassilev and colleagues.30 Both
5da and nutlin-3 could decrease MDM2 protein in T24 cells, but almost no effect on
the expression of p53. These results suggested that 5da exhibited toxicity for
cancerous was predominantly due to p53 activation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Generally speaking, p53 activation is a common response to distinct cellular
stresses, which induces cell cycle arrest or apoptosis by governing the downstream
signaling molecules.31 Because p21 is the direct target of p53 regulatory circuit, we
investigated the cyclinE-CDK2 complexes, cyclinD-CDK4 complexes and
cyclin-dependent kinase (CDK) inhibitor p21 variance due to their functions in cell
cycle progression. Cyclin E binds to CDK2, which is required for the transition from
G1 to S phase during cell division.32 Our results showed that 5da could induce
significant increase of cyclinE-CDK2 complexes to promote G1 to S phase transition
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 56
1
2
3
4
5
6
7
8
9
(Figure 2I). However, nearly no change was found in the expression of cyclin D and
CDK4. Instead, 5da could up-regulate p21 expression in a dose-dependent manner
(Figure 2I). These results suggested that S/G2 arrest induced by 5da resulted from the
inhibition of CDK activity by p21 rather than the expression reduction of cyclin D and
CDK4.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5da Induces Cell Apoptosis through the Intrinsic Apoptotic Pathway. To gain
further insight into the mechanisms of tumor suppression activity of 5da, we wanted
to confirm firstly whether the sub-G1 phase cells induced by 5da were due to
apoptosis. The 5da-treated cells were therefore investigated with PI and
FITC-Annexin V staining for apoptosis identification. As depicted in Figure 3A, cells
treated by 5da displayed obvious cell apoptosis in T24 cells in a dose-dependent
manner. Compared to vehicle-treated cells, 5da induced 4.22% (1 μM), 26.4% (5 μM)
and 29.4% (10 μM) of apoptotic cells, respectively. To confirm the proapoptotic effect
of 5da in T24 cells, cell apoptosis with activated caspase-3 was also studied. As
shown in Figure 3B, the peaks for cells with activated caspase-3 were gradually
increased in the 5da-treated cells compared with the control group. The proportions of
caspase-3 activated cells after the 5da treatment were 7.0% (1 μM), 17.6% (5 μM)
and 50.0% (10 μM) in the T24 cells. Furthermore, after the treatment of 5da,
Apoptosis-inducing Factor (AIF) was released from mitochondria and translocated to
the nucleus to trigger apoptosis (Figure 3C).
The observations prompted us to verify which death effectors in the mitochondria
were dynamic participants in the death signaling, because AIF is a mitochondrial
10
ACS Paragon Plus Environment

Page 11 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
protein.33 Bcl-2 family proteins were firstly chosen to examine how the abundance of
likely candidate proteins varied after 5da treatment. As shown in Figure 3D, the most
increase was in the levels of Bim, Bax, and Bak, while Bcl-xl, Bcl-2 showed
insignificant change. These results collectively indicated that 5da-induced cell
apoptosis were mainly through mitochondrial-mediated intrinsic apoptotic pathway.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3
5da Induces Conformational Activation of Bak for Cell Apoptosis by
Mitochondrial Accumulation of Phospho-p53. Conformational activation of Bak
characterized by N-terminal exposure is a distinct marker of the sensitized
mitochondrial apoptotic pathway.34,35 Having confirmed the activated intrinsic
apoptotic pathway, we sought to find out whether 5da could conformationally activate
Bak, thus defining a framework to understand the mechanism for apoptotic signals
cascading. Immunohistochemistry method was adopted to examine the Bak activation.
As depicted in Figure 4A, almost no activated Bak could be observed in the control
group, reflecting the fact that Bak without activation could not play the role of death
effector even in the case of high abundance. However, after the treatment by 5da,
conformationally altered Bak could be captured by N-terminal Bak antibody, and such
phenomenon was more obvious when the drug concentration increased. The results
indicated that 5da could conformationally activate Bak for cell apoptosis.
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 56
1
2
3
4
5
6
7
8
9
Conformational activation of Bak is a dissociation step from antiapoptotic Bcl-2
family members to form Bak homodimers, eventually leading to generate the pore
forming activity.36 Previous studies have shown that antiapoptotic Bcl-2 family
member Bcl-xl controls the conformational status of Bak by way of forming
Bak–Bcl-xl complex.34,35 That fact raised the possibility that 5da-induced Bak
activation may be due to disrupting the Bak–Bcl-xl interaction. To answer such a
question, coimmunoprecipitation analysis was adopted to study the two proteins of
interests. As expected, 5da could disrupt the Bak–Bcl-xl interaction (Figure 4C) and
without affecting the expression of Bcl-xl (Figure 3D).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Having established Bak activation by 5da-induced Bak–Bcl-xl complex
perturbation, what interested us most was how 5da predominantly affected cells to
transfer proapoptotic stimuli. 5da has been confirmed to be a p53 activator in our
study. We found that 5da did not affect p53 expression, but significantly elevated p53
phosphorylation (Figure 2G), and phospho-p53 mainly accumulated in mitochondria
(Figure 4B). Previous studies have shown that activated p53 always was translocated
into mitochondria to trigger cell apoptosis through the transcription-independent p53
pathways.36,37 It is therefore quite reasonable for us to study the potential role of p53
in 5da-induced Bak activation. To analyze p53’s ability to perturb Bak–Bcl-xl
interaction after 5da treatment, we coimmunoprecipitated phospho-p53 with Bcl-xl
and Bcl-xl with Bak in T24 cells. As shown in Figure 4C, p53 exhibited direct
interaction and high affinity for Bcl-xl after 5da treatment for 24 h, which was
consistent with the previous report,38 while Bak dissociated from Bak–Bcl-xl complex.
12
ACS Paragon Plus Environment

Page 13 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
The results suggested that 5da-induced mitochondrial accumulation of phospho-p53
6
7
promoted Bak activation even when it stayed bound to Bcl-xl.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4
5da Shows Potential Anticancer Potency in Vivo with Low Toxicity. The
potential clinical utility of 5da was finally examined in HepG2 xenograft tumors in
nude mice. In the HepG2 xenograft models, mice with tumors at the volume of
80200 mm3 were randomized into treatment and vehicle control groups (n=6/group),
and treated via ip injection of 5da or HCPT, respectively, every other day. The
tumor-bearing mice treated by 5da (6 or 12 mg/kg/2days) showed significant tumor
growth inhibition over the treatment period compared with those in vehicle-treated,
tumor-bearing mice (Figure 5A, B and C). The results of tumor growth inhibition
(TGI) identical to vehicle control group reached to 43.6% (5da, 6 mg/kg/2days),
61.3% (5da, 12 mg/kg/2days) and 71.6% (HCPT, 6 mg/kg/2days) respectively
(P<0.001). In all experiments, except for a slight body weight decrease in
HCPT-treated animals, no differences were found between the control group and
5da-treated animals in clinical signs of toxicity and food consumption (Figure 5D).
To consistent with the in vitro findings, we also checked the expression level of p53
and Bak as well as anticancer activity in the T24 xenograft models. As depicted in
Figure 5E, the results of tumor growth inhibition (TGI) identical to vehicle control
group reached to 44.8% (5da, 6 mg/kg/2days) and 54.2% (5da, 12 mg/kg/2days)
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 56
1
2
3
4
5
6
7
8
9
respectively (P<0.001). Consistent with the in vitro findings, 5da induced p53
activation in vivo along with little impact on p53 expression (Figure 5F). However,
high dose administration (5da, 12 mg/kg/2days) could significantly decrease the Bak
expression in vivo. This may be related to too much dosage, as in vitro studies (Figure
3D), the Bak was also down-regulated in T24 cells after treatment with 5da (10 μM).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5
DISCUSSION AND CONCLUSIONS
In this study, a new series of p53 activators based on the phthalazino[1,2-b]-
-quinazolinone motif have been designed and synthesized. Some of the analogs
exhibit promising anti-cancer activities. Our results indicated that 5da could
effectively suppress cell proliferation in T24 cancer cells and obviously changed the
cell size. Moreover, we verified that the antitumor effect of 5da is likely due to the
induction of apoptosis by regulating the related targets based on
mitochondria-targeted accumulation of phospho-p53.
Cell cycle arrest is a common response to p53 activation.39,40 In our effort to
elaborate its molecular mechanism, we found that 5da could induce significant cell
cycle arrest in S/G2 phase after treatment 24 h with consistent variance in G2-related
target p21. p21 is a direct target of p53-induced transcriptional program.41 5da could
up-regulate p21 expression for cell cycle response but had no influence on cyclin D
and CDK4, two other functional proteins in S and G2 phase progression.
14
ACS Paragon Plus Environment

Page 15 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
We further examined the cell apoptosis evoked by 5da in T24 cells. We found that
5da induced cell apoptosis identified by membrane blebbing, caspase-3 activation and
AIF release from mitochondria. Mitochondria-mediated intrinsic apoptotic pathway is
always involved in small molecule compound-induced cell apoptosis.42 With the
observations of cell apoptosis and mitochondrial special protein AIF release, we
verified which death effectors in the mitochondria were dynamic participants in the
death signaling and found the increase of proapoptotic proteins in Bax, Bim and Bak.
Conformational activation of Bak is a distinct marker of the sensitized
mitochondrial apoptotic pathway.34,35 Here we found that 5da-induced mitochondrial
accumulation of phospho-p53 conformationally activated Bak by reorganizing the
Bak–Bcl-xl complex. Bcl-xl controls conformational status of Bak by physical
interaction,35 conformational activation of Bak is a dissociation step from Bcl-xl to
form Bak homodimers.36 Coimmunoprecipitation analysis suggested that
phospho-p53 exhibited direct interaction and high affinity for Bcl-xl after 5da
treatment, while Bak passively dissociated from Bak–Bcl-xl complex.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Although 5da was proven valuable as a chemotherapeutic agent for BC therapy
with the relatively clear molecular mechanism, the exact mechanism of how 5da
interferes with p53 activation is still unclear. Further study should be carried out to
verify if 5da interacts with p53 directly or indirectly. Moreover, pharmacodynamic
evaluation in vitro and in vivo suggested that 5da was more potent than HCPT in vitro,
but less potent in vivo. The significant difference of activity between in vitro and in
vivo indicated bioavailability of 5da may be an important factor to affect its biological
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 56
1
2
3
4
5
activity, which restricted its clinical application.
6
7
8
9
In conclusion, we present here a small-molecule platform that enables a switch to
trigger cell apoptosis by manipulating p53 apoptotic signaling pathway, emphasized
the molecular regulatory networks for pharmacological characterization. These results
provide strong evidences for the preclinical efficacy of 5da as a promising anticancer
agent in BC therapy or an efficient adjuvant in p53 restoration-mediated anticancer
therapy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
Materials. All reagents were purchased from commercial sources and were used
without further purification unless otherwise noted. All reactions were carried out
under an atmosphere of dry nitrogen. Melting points were recorded on WRS-IA
apparatus without correction. NMR spectra were recorded in DMSO-d6, CDCl3 or
CD3OD on Bruker Advance (500 or 400 MHz) with TMS as internal standard. HRMS
were measured in EI or ESI mode and the mass analyzer of the HRMS was TOF.
Flash column chromatography was performed on silica gel (200-300 mesh).
Propidium iodide (PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) were purchased from Sigma (St. Louis, MO). Anti-phospho p53
(s392), anti-p53, anti-p21, anti-Bim, anti-Bcl-2, anti-Bcl-xl, anti-AIF, anti-lamin B,
anti-cox IV, anti-actin and anti-Bax antibodies were purchased from Abcam
(Cambridge, MA). Anti-Bak antibody and Caspase-3 activity fluorescence detection
kit were purchased from Calbiochem. Anti-rabbit and –mouse secondary antibodies
16
ACS Paragon Plus Environment

Page 17 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
6
7
8
9
All synthetic compounds 5 were analyzed by HPLC (254 nm). The system was
equipped with a C18 5µm SHIMADZU-GL ODS-3 column (4.6*250 mm) with a flow
rate of 0.7 mL/min. HPLC analysis showed that the purity of all compounds
were >95%, and their spectra could be found in Supporting Information. Compounds
5 were stored at 20 °C at a concentration of 10 mM in dimethyl sulfoxide (DMSO).
General procedure for synthesis of 10-substitued 6H-phthalazino[1,2-b]
quinazolin-5,8-diones (3). Phthalic anhydride (1.48 g, 0.01 mol) and hydrazide 2
(0.01 mol) in glycol (15 mL) was heated in 150 oC oil bath for 2 h until the reaction
completed (monitored by TLC). When the mixture was cooled, the solid was
vacuum filtered and washed with ethyl acetate (10 mL×2) to give pale yellow solid 3
which were used in next step without further purification. 3a: yield 89%, mp 288 °C
(dec.); 3b: yield 82%, mp 193 °C (dec.); 3c: yield 64%, mp 166-167 °C; 3d: yield
83%, mp 202 °C (dec.); 3e: yield 92%, mp 266 °C (dec.).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General procedure for synthesis of 10-substitued 5-chlorophthalazino[1,2-b]
quinazolin-8-ones (4). 3 (0.01 mol) was mixture with redistilled POCl3 (10 mL) and
o
heated in 90 C oil bath under nitrogen atmosphere for about 6 h. After completed
(monitored by TLC), the excess POCl3 was removed by reduced distillation and ice
water was poured in. The precipitate was filtered off, washing successively with water
and methanol to give pale yellow solid 4 which were used directly in next step. 4a:
yield 89%, mp 156-158 °C (dec.); 4b: yield 82%, mp 143-145 °C; 4c: yield 64%, mp
187-189 °C (dec.); 4d: yield 83%, mp 202 °C (dec.); 4e: yield 92%, mp 163-166 °C.
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 56
1
2
3
4
5
6
7
General procedure for synthesis of 5, 10-disubstitued phthalazino[1,2-b]-
-quinazolin-8-ones (5). 4 (5 mmol) and amine (10 mmol) in toluene (10 mL) was
8
9
o
heated in 90 C oil bath for 12 h until the reaction completed (monitored by TLC).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Then the solvent was removed under reduced pressure and the residue was purified by
column chromatography on silica gel (eluent: EtOAc/DCM/methanol = 9/3/1) to
afford 5 as pale yellow or white solid.
5-(2-Dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one
(5aa).
Following the general procedure, 4a (1.41 g, 5 mmol) and 2-dimethylamino-
-ethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a
pale yellow solid (1.05 g, 63 % yield), mp 219~221 °C; 1H (CD3OD) δ 8.74 (dd, 1H, J
= 6.2, 3.0 Hz), 8.23 (dd, 1H, J = 8.1, 0.9 Hz), 7.94 (dd, 1H, J = 6.1, 3.0 Hz),
7.77~7.69 (m, 3H), 7.67 (d, 1H, J = 7.7 Hz), 7.45~7.35 (m, 1H), 3.75 (t, 2H, J = 6.4
13
Hz), 2.79 (t, 2H, J = 6.4 Hz), 2.41 (s, 6H). C NMR (125 MHz, CD3OD) δ 159.0,
149.2, 146.2, 142.8, 133.6, 132.5, 131.8, 128.6, 126.9, 126.3, 126.2, 125.5, 122.1,
121.4, 119.4, 57.5, 44.1, 38.6. HRMS (ESI) m/z: calcd for C19H20N5O [M+H]+
334.1668; found 334.1660. RP-C18-HPLC: 97.342%, tR = 17.897 min.
5-(3-Dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one
(5ab).
Following the general procedure, 4a (1.41 g, 5 mmol) and 3-dimethylamino-
-propylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a
pale yellow solid (0.96 g, 55% yield), mp 158~159 °C; 1H NMR (500 MHz, CDCl3) δ
8.88 (d, J = 7.6 Hz, 1H), 8.24 (dd, J = 7.6, 3.5 Hz, 2H), 7.98~7.90 (m, 2H), 7.83 (t, J
= 7.5 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.68 (s, 1H), 7.51 (t, J = 7.4 Hz, 1H), 3.51 (dd,
18
ACS Paragon Plus Environment

Page 19 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
J = 12.5, 6.6 Hz, 2H), 2.38 (t, J = 6.8 Hz, 2H), 2.20 (s, 6H,), 1.90 (t, J = 7.0, 2H); 13C
NMR (125 MHz, DMSO-d6) δ 157.8, 149.0, 146.3, 143.6, 134.2, 133.4, 132.7, 129.0,
127.5, 127.1, 126.7, 126.0, 123.3, 122.0, 120.3, 57.7, 45.7, 40.5, 26.2. HRMS (ESI)
m/z: calcd for C20H22N5O [M+H]+ 348.1824; found 348.1816. RP-C18-HPLC:
95.985%, tR = 21.009 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-(3-Morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin-8-one
(5ac).
Following the general procedure, 4a (1.41 g, 5 mmol) and 3-morpholinopropan-
-1-amine (1.44 g, 10 mmol) were used, and the desired product was obtained as a pale
1
yellow solid (1.57 g, 81% yield), mp 158~162 °C; H NMR (500 MHz, DMSO-d6) δ
8.86 (dd, J = 7.5, 1.2 Hz, 1H), 8.25 (dd, J = 19.9, 7.7 Hz, 2H), 7.96~7.89 (m, 2H),
7.81 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.60 (s, 1H), 7.49 (t, J = 7.5 Hz,
1H), 3.74 (t, J = 4.5 Hz, 2H), 3.62 (t, J = 4.1 Hz, 4H), 3.00 (t, J = 4.3 Hz, 2H), 2.42 (t,
13
J = 6.8 Hz, 4H), 1.92 (m, 5.5 Hz, 2H); C NMR (125 MHz, DMSO-d6) δ 157.7,
149.0, 146.3, 143.6, 134.1, 133.3, 132.7, 129.0, 127.5, 127.0, 126.7, 126.0, 123.4,
122.0, 120.3, 66.6, 56.8, 53.8, 39.5, 25.1. HRMS (ESI) m/z: calcd for C22H24N5O2
[M+H]+ 390.1930; found 390.1922. RP-C18-HPLC: 99.293%, tR = 18.011 min.
5-(Morpholin-4-yl)phthalazino[1,2-b]quinazolin-8-one (5ad). Following the
general procedure, 4a (1.41 g, 5 mmol) and morpholine (0.87 g, 10 mmol) were used,
and the desired product was obtained as a pale yellow solid (1.49 g, 90% yield), mp
221~222 °C; 1H NMR (500 MHz, CDCl3) δ 8.98 (dd, J = 7.9, 1.4 Hz, 1H), 8.46 (dd, J
= 8.0, 0.8 Hz, 1H), 7.93 (dd, J = 7.7, 1.3 Hz, 1H), 7.85~7.77 (m, 4H), 7.49 (ddd, J =
8.0, 6.7, 1.5 Hz, 1H), 3.98 (t, J = 4.6 Hz, 4H), 3.50 (t, J = 4.6 Hz, 4H); 13C NMR (125
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 56
1
2
3
4
5
6
7
8
9
MHz, CDCl3) δ 158.7, 154.5, 146.6, 143.6, 134.4, 132.5, 132.4, 130.9, 127.7, 127.5,
127.3, 126.2, 125.2, 122.7, 120.6, 66.8, 51.5. HRMS (ESI) m/z: calcd for C19H17N4O2
[M+H]+ 333.1352; found 333.1348. RP-C18-HPLC: 98.812%, tR = 30.408 min.
5-(4-Methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5ae). Following
the general procedure, 4a (1.41 g, 5 mmol) and 1-methylpiperazine (1.00 g, 10 mmol)
were used, and the desired product was obtained as a pale yellow solid (1.21 g, 70%
yield), mp 197~198 °C; 1H NMR (500 MHz, CDCl3) δ 8.98 (dd, J = 7.8, 1.4 Hz, 1H),
8.47 (dd, J = 8.0, 1.2 Hz, 1H), 7.93 (dd, J = 7.8, 1.2 Hz, 1H), 7.84~7.77 (m, 4H), 7.49
(ddd, J = 8.1, 6.8, 1.4 Hz, 1H), 3.55 (s, 4H), 2.72 (s, 4H), 2.41 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 158.8, 154.5, 146.7, 143.7, 134.3, 132.3, 132.3, 130.9, 127.7, 127.5,
127.3, 126.1, 125.4, 123.0, 120.7, 54.9, 50.8, 46.3. HRMS (ESI) m/z: calcd for
C20H20N5O [M+H]+ 346.1668; found 346.1660. RP-C18-HPLC: 99.696%, tR = 22.346
min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10-Fluoro-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one
(5ba). Following the general procedure, 4b (1.50 g, 5 mmol) and 2-dimethylamino-
-ethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a
pale yellow solid (1.51 g, 86% yield), mp 125~226 °C; 1H NMR (500 MHz, CD3OD)
δ 8.66 (dd, J = 6.1, 3.3 Hz, 1H), 7.95 (dd, J = 5.8, 3.3 Hz, 1H), 7.82 (dd, J = 6.1, 3.2
Hz, 2H), 7.74 (dd, J = 8.7, 2.9 Hz, 1H), 7.65 (dd, J = 9.0, 4.9 Hz, 1H), 7.51 (td, J =
13
8.6, 2.9 Hz, 1H), 3.82 (t, J = 5.7 Hz, 2H), 3.13 (t, J = 5.5 Hz, 2H), 2.76 (s, 6H); C
NMR (125 MHz, CD3OD) δ 162.5, 160.5, 159.5, 151.1, 144.3, 143.1, 134.0, 133.6,
131.2, 129.6, 127.5, 123.6, 122.3, 121.4, 111.6, 59.8, 45.1, 39.8. HRMS (ESI) m/z:
20
ACS Paragon Plus Environment

Page 21 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
calcd for C19H19FN5O [M+H]+ 352.1574; found 352.1567. RP-C18-HPLC: 99.268%,
tR = 18.222 min.
7
8
9
10-Fluoro-5-(2-dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one
(5bb). Following the general procedure, 4b (1.50 g, 5 mmol) and 3-dimethylamino-
-propylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a
pale yellow solid (1.61 g, 88% yield), mp 198~200 °C; 1H NMR (500 MHz, CD3OD)
δ 8.62~8.57 (m, 1H), 7.86~7.82 (m, 1H), 7.75~7.70 (m, 2H), 7.69 (dd, J = 8.8, 2.9 Hz,
1H), 7.58 (dd, J = 9.0, 4.9 Hz, 1H), 7.43 (td, J = 8.6, 3.0 Hz, 1H), 3.60 (t, J = 6.7 Hz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
2H), 2.76 (t, J = 7.0 Hz, 2H), 2.53 (s, 6H), 2.07~1.98 (m, 2H); C NMR (125 MHz,
CD3OD) δ 162.5, 160.5, 159.4, 151.4, 144.3, 143.4, 133.9, 131.1, 129.7, 127.5, 124.0,
123.4, 122.6, 121.6, 111.6, 57.6, 44.9, 40.6, 27.1. HRMS (ESI) m/z: calcd for
C20H21FN5O [M+H]+ 366.1730; found 366.1725. RP-C18-HPLC: 95.914%, tR =
18.265 min.
10-Fluoro-5-3-morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin-8-one
(5bc). Following the general procedure, 4b (1.50 g, 5 mmol) and 3-morpholino-
-propan-1-amine (1.44 g, 10 mmol) were used, and the desired product was obtained
1
as a pale yellow solid (1.67 g, 82% yield), mp 232~233 °C; H NMR (500 MHz,
CDCl3) δ 8.96 (dd, J = 7.3, 2.0 Hz, 1H), 8.05 (dd, J = 8.7, 3.0 Hz, 1H), 7.86 (dd, J =
6.3, 2.6 Hz, 1H), 7.83~7.77 (m, 3H), 7.54 (s, 1H), 7.51~7.46 (m, 1H), 3.84 (t, J = 4.5
Hz, 4H), 3.79 (dd, J = 10.3, 5.6 Hz, 2H), 2.67 (t, J = 5.5 Hz, 2H), 2.61 (s, 4H),
2.00~1.91 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 161.4, 159.4, 158.1, 149.2, 143.2,
142.7, 132.4, 132.1, 129.8, 129.5, 127.3, 122.9, 121.9, 121.6, 111.9, 67.1, 59.5, 54.1,
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 56
1
2
3
4
5
6
43.3, 23.3. HRMS (ESI) m/z: calcd for C22H23FN5O2 [M+H]+ 408.1836; found
408.1830. RP-C18-HPLC: 98.134%, tR = 18.534 min.
7
8
9
10-Fluoro-5-(morpholin-4-yl)phthalazino[1,2-b]quinazolin-8-one
(5bd).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Following the general procedure, 4b (1.50 g, 5 mmol) and morpholine (0.87 g, 10
mmol) were used, and the desired product was obtained as a pale yellow solid (1.40 g,
80% yield), mp 277~278 °C; 1H NMR (500 MHz, CDCl3) δ 8.94 (dd, J = 7.7, 1.6 Hz,
1H), 8.06 (dd, J = 8.6, 3.0 Hz, 1H), 7.94 (dd, J = 7.8, 1.4 Hz, 1H), 7.86~7.78 (m, 3H),
13
7.54~7.49 (m, 1H), 3.98 (t, J = 4.6 Hz, 4H), 3.50 (t, J = 4.6 Hz, 4H); C NMR (125
MHz, CDCl3) δ 161.5, 159.6, 158.1, 154.8, 143.4, 143.0, 132.6, 130.8, 130.0, 127.2,
125.3, 123.4, 122.6, 121.7, 112.2, 66.7, 51.5. HRMS (ESI) m/z: calcd for
C19H16FN4O2 [M+H]+ 351.1257; found 351.1254. RP-C18-HPLC: 97.205%, tR =
21.819 min.
10-Fluoro-5-(4-methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5be).
Following the general procedure, 4b (1.50 g, 5 mmol) and 1-methylpiperazine (1.00 g,
10 mmol) were used, and the desired product was obtained as a pale yellow solid
(1.45 g, yield 80%), mp 208~209 °C; 1H NMR (500 MHz, CDCl3) δ 8.94 (dd, J = 7.7,
1.2 Hz, 1H), 8.06 (dd, J = 8.6, 2.9 Hz, 1H), 7.93 (dd, J = 6.5, 1.2 Hz, 1H), 7.85~7.81
13
(m, 3H), 7.55~7.48 (m, 1H), 3.56 (s, 4H,), 2.73 (s, 4H), 2.42 (s, 3H); C NMR (125
MHz, CDCl3) δ 161.5, 159.5, 158.1, 154.8, 143.4, 143.1, 132.4, 130.8, 129.9, 127.2,
125.4, 123.4, 122.8, 121.7, 112.2, 54.7, 50.7, 46.2. HRMS (ESI) m/z: calcd for
C20H19FN5O [M+H]+ 364.1574; found 364.1567. RP-C18-HPLC: 95.026%, tR =
25.709 min.
22
ACS Paragon Plus Environment

Page 23 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10-Chloro-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one
(5ca). Following the general procedure, 4c (1.58 g, 5 mmol) and 2-dimethylamino-
-ethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a
pale yellow solid (0.81 g, 44% yield), mp 294~297 °C; 1H NMR (500 MHz, CD3OD)
δ 8.67~8.64 (m, 1H), 7.98 (dd, J = 5.6, 2.3 Hz, 1H), 7.95 (d, J = 2.4 Hz, 1H),
7.90~7.83 (m, 2H), 7.63 (dd, J = 8.8, 2.4 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 3.81 (t, J
= 4.4, 2H), 3.49 (t, J = 4.4, 2H), 3.09 (s, 6H); 13C NMR (125 MHz, CD3OD) δ 158.0,
150.6, 144.6, 142.1, 134.4, 133.2, 133.1, 131.5, 129.1, 128.2, 126.3, 125.1, 122.9,
120.6, 119.5, 60.0, 43.0, 38.2. HRMS (ESI) m/z: calcd for C19H19ClN5O [M+H]+
368.1278; found 368.1273. RP-C18-HPLC: 98.536%, tR = 19.701 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10-Chloro-5-(2-dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one
(5cb). Following the general procedure, 4c (1.58 g, 5 mmol) and 3-dimethylamino-
-propylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a
pale yellow solid (1.45 g, 76% yield), mp 224~226 °C; 1H NMR (500 MHz, CD3OD)
δ 8.78~8.75 (m, 1H), 8.11 (d, J = 2.3 Hz, 1H), 8.09~8.04 (m, 1H), 7.93~7.84 (m, 2H),
7.69 (dd, J = 8.8, 2.4 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 3.74 (t, J = 6.1 Hz, 2H), 3.21
(t, J = 6.3 Hz, 2H), 2.93 (s, 6H), 2.29~2.15 (m, 2H); 13C NMR (500 MHz, CD3OD) δ
158.0, 151.6, 144.7, 142.6, 134.3, 133.1, 132.7, 131.3, 129.1, 128.4, 126.4, 125.2,
122.6, 121.1, 119.8, 53.6, 42.2, 37.5, 25.7. HRMS (ESI) m/z: calcd for C20H21ClN5O
[M+H]+ 382.1435; found 382.1431. RP-C18-HPLC: 96.601%, tR = 19.947 min.
10-Chloro-5-(3-morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin-8-
-one (5cc). Following the general procedure, 4c (1.58 g, 5 mmol) and 3-morpholino-
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 56
1
2
3
4
5
-propan-1-amine (1.44 g, 10 mmol) were used, and the desired product was obtained
6
7
1
as a pale yellow solid (1.70 g, 80% yield), mp 231~233 °C; H NMR (500 MHz,
8
9
CDCl3) δ 8.98~8.93 (m, 1H), 8.38 (d, J = 2.4 Hz, 1H), 7.90~7.85 (m, 2H), 7.83~7.79
(m, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.66 (dd, J = 8.8, 2.4 Hz, 1H), 7.55 (s, 1H), 3.85 (t,
J = 4.6 Hz, 4H), 3.79 (dd, J = 10.3, 5.7 Hz, 2H), 2.70~2.67 (m, 2H), 2.62 (s, 4H),
2.00~1.93 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 157.0, 148.5, 144.2, 142.8, 133.5,
131.8, 131.4, 130.7, 128.6, 128.3, 126.7, 125.8, 121.2, 121.1, 120.6, 66.3, 58.7, 53.4,
42.4, 22.5. HRMS (ESI) m/z: calcd for C22H23ClN5O2 [M+H]+ 424.1540; found
424.1533. RP-C18-HPLC: 97.240%, tR = 20.421 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10-Chloro-5-(morpholin-4-yl)phthalazino[1,2-b]quinazolin-8-one
(5cd).
Following the general procedure, 4c (1.58 g, 5 mmol) and morpholine (0.87 g, 10
mmol) were used, and the desired product was obtained as a pale yellow solid (1.71 g,
93% yield), mp 258~259 °C; 1H NMR (500 MHz, CDCl3) δ 8.99 (dd, J = 7.7, 1.4 Hz,
1H), 8.42 (d, J = 2.4 Hz, 1H), 7.97 (dd, J = 7.2, 1.8 Hz, 1H), 7.86~7.82 (m, 2H), 7.79
(d, J = 8.8 Hz, 1H), 7.73 (dd, J = 8.8, 2.4 Hz, 1H), 3.99 (t, J = 4.6, 4H), 3.52 (t, J =
13
4.5, 4H); C NMR (125 MHz, CDCl3) δ 157.6, 154.7, 145.0, 143.7, 134.8, 132.5,
132.5, 131.8, 130.6, 129.0, 127.3, 126.7, 125.1, 122.6, 121.4, 66.6, 51.4. HRMS (ESI)
m/z: calcd for C19H16ClN4O2 [M+H]+ 367.0962; found 367.0937. RP-C18-HPLC:
97.101%, tR = 34.262 min.
10-Chloro-5-(4-methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5ce).
Following the general procedure, 4c (1.58 g, 5 mmol) and 1-methyl-piperazine (1.00 g,
10 mmol) were used, and the desired product was obtained as a pale yellow solid
24
ACS Paragon Plus Environment

Page 25 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
1
(1.71 g, 90% yield), mp 206~208 °C; H NMR (500 MHz, CDCl3) δ 8.37 (d, J = 2.4
6
7
8
9
Hz, 1H), 7.92 (dd, J = 5.6, 2.1 Hz, 1H), 7.82~7.79 (m, 2H), 7.74 (d, J = 8.7 Hz, 1H),
7.69 (dd, J = 8.7, 2.4 Hz, 1H), 3.54 (s, 4H), 2.71 (s, 4H), 2.41 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 157.8, 154.8, 145.1, 143.9, 134.8, 132.5, 132.4, 131.8, 130.7, 129.1,
127.3, 126.8, 125.4, 122.9, 121.5, 54.9, 50.8, 46.3. HRMS (ESI) m/z: calcd for
C20H19ClN5O [M+H]+ 380.1278; found 380.1270. RP-C18-HPLC: 98.399%, tR =
19.247 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10-Bromo-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one
(5da). Following the general procedure, 4d (1.80 g, 5 mmol) and 2-dimethylamino-
-ethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a
pale yellow solid (1.40 g, 68% yield), mp 141~146 °C; 1H NMR (500 MHz, CD3OD)
δ 8.43 (d, J = 7.6 Hz, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H),
7.69~7.62 (m, 2H), 7.56 (dd, J = 8.6, 2.1 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 3.69 (t, J
= 5.8 Hz, 2H), 2.93 (t, J = 5.8 Hz, 2H), 2.57 (s, 6H); 13C NMR (125 MHz, CD3OD) δ
161.4, 153.4, 148.5, 146.6, 140.4, 136.6, 135.9, 132.7, 132.3, 131.9, 130.1, 126.1,
124.9, 124.2, 122.5, 61.9, 47.9, 42.4. HRMS (ESI) m/z: calcd for C19H19BrN5O
[M+H]+ 412.0773; found 412.0769. RP-C18-HPLC: 98.463%, tR = 13.635 min.
10-Bromo-5-(2-dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one
(5db). Following the general procedure, 4d (1.80 g, 5 mmol) and 3-dimethylamino-
-propylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a
1
pale yellow solid (1.60 g, 75% yield), mp 215~217 °C; H NMR (500 MHz,
DMSO-d6) δ 8.84 (d, J = 7.5 Hz, 1H), 8.27 (s, 1H), 8.22 (d, J = 7.5 Hz, 1H),
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 56
1
2
3
4
5
7.98~7.88 (m, 3H), 7.70 (d, J = 8.7 Hz, 2H), 3.51 (dd, J = 11.3, 5.9 Hz, 2H), 2.39 (t, J
6
7
13
= 6.5 Hz, 2H), 2.21 (s, 6H), 1.94~1.85 (m, 2H); C NMR (125 MHz, DMSO-d6) δ
8
9
156.64, 149.2, 145.2, 144.1, 136.9, 133.6, 132.7, 129.9, 128.9, 128.8, 126.8, 123.3,
122.0, 121.7, 118.2, 57.8, 56.5, 45.7, 26.2. HRMS (ESI) m/z: calcd for C20H21BrN5O
[M+H]+ 426.0929; found 426.0922. RP-C18-HPLC: 95.188%, tR = 23.679 min.
10-Bromo-5(3-morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin-8-one
(5dc). Following the general procedure, 4d (1.80 g, 5 mmol) and 3-morpholino-
-propan-1-amine (1.44 g, 10 mmol) were used, and the desired product was obtained
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
as a pale yellow solid (1.90 g, 81% yield), mp 236~238 °C; H NMR (500 MHz,
CDCl3) δ 8.98~8.92 (m, 1H), 8.53 (d, J = 2.2 Hz, 1H), 7.86~7.81 (m, 3H), 7.79 (dd, J
= 9.0, 2.3 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.57 (s, 1H), 3.84 (t, J = 4.5 Hz, 4H),
3.78 (dd, J = 9.7, 4.9 Hz, 2H), 2.68~2.64 (m, 2H), 2.60 (s, 4H), 1.98~1.89 (m, 2H);
13C NMR (125 MHz, CDCl3) δ 157.5, 149.1, 145.1, 143.5, 136.7, 132.4, 132.0, 129.6,
129.2, 129.0, 127.3, 121.8, 121.6, 119.0, 67.1, 59.5, 54.0, 43.3, 23.1. HRMS (ESI)
m/z: calcd for C22H23BrN5O2, [M+H]+ 468.1035 found 468.1030. RP-C18-HPLC:
98.748%, tR = 20.902 min.
10-Bromo-5-(morpholin-4-yl)phthalazino[1,2-b]quinazolin-8-one
(5dd).
Following the general procedure, 4d (1.80 g, 5 mmol) and morpholine (0.87 g, 10
mmol) were used, and the desired product was obtained as a pale yellow solid (1.40 g,
68% yield), mp 254~255 °C; 1H NMR (500 MHz, CDCl3) δ 8.99 (dd, J = 7.7, 1.7 Hz,
1H), 8.58 (d, J = 2.3 Hz, 1H), 7.97 (dd, J = 6.2, 1.5 Hz, 1H), 7.89~7.84 (m, 3H), 7.72
(d, J = 8.7 Hz, 1H), 3.99 (t, J = 4.6 Hz, 4H), 3.52 (t, J = 4.6 Hz, 4H); 13C NMR (125
26
ACS Paragon Plus Environment

Page 27 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
MHz, CDCl3) δ 156.7, 153.8, 144.5, 143.0, 136.6, 131.7, 131.7, 129.8, 129.0, 128.4,
126.5, 124.3, 121.8, 120.9, 118.7, 65.8, 50.5. HRMS (ESI) m/z: calcd for
C19H16BrN4O2 [M+H]+ 411.0457; found 411.0453. RP-C18-HPLC: 98.795%, tR =
33.722 min.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10-Bromo-5-(4-methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5de).
Following the general procedure, 4d (1.80 g, 5 mmol) and 1-methylpiperazine (1.00 g,
10 mmol) were used, and the desired product was obtained as a pale yellow solid
(1.21 g, 57% yield), mp 220~221 °C; 1H NMR (500 MHz, CDCl3) δ 8.94 (dd, J = 7.3,
2.0 Hz, 1H), 8.55 (d, J = 2.3 Hz, 1H), 7.93 (dd, J = 7.2, 2.0 Hz, 1H), 7.85~7.80 (m,
3H), 7.68 (d, J = 8.7 Hz, 1H), 3.56 (s, 4H), 2.73 (s, 4H,), 2.42 (s, 3H). 13C NMR (125
MHz, CDCl3) δ 157.6, 154.8, 145.5, 144.1, 137.5, 132.6, 132.5, 130.7, 130.0, 129.3,
127.3, 125.5, 123.0, 121.9, 119.5, 54.8, 50.8, 46.3. HRMS (ESI) m/z: calcd for
C20H19BrN5O [M+H]+ 424.0773; found 424.0768. RP-C18-HPLC: 98.476%, tR =
19.826 min.
10-Methyl-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one
(5ea). Following the general procedure, 4e (1.48 g, 5 mmol) and 2-dimethylamino-
-ethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a
pale yellow solid (1.18 g, 68% yield), mp 124 °C (dec.); 1H NMR (500 MHz, CD3OD)
δ 8.33~8.30 (m, 1H), 7.66~7.62 (m, 1H), 7.57 (s, 1H), 7.55~7.51 (m, 2H), 7.22 (dd, J
= 8.4, 1.7 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 3.61 (t, J = 5.6 Hz, 2H), 2.99 (t, J = 5.3
13
Hz, 2H), 2.63 (s, 6H), 2.24 (s, 3H). C NMR (125 MHz, CD3OD) δ 162.3, 153.2,
147.7, 145.1, 139.7, 139.0, 136.0, 135.8, 132.1, 130.6, 129.8, 129.0, 126.0, 124.5,
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 56
1
2
3
4
5
6
122.4, 62.4, 47.7, 42.3, 24.1. HRMS (ESI) m/z: calcd for C20H22N5O [M+H]+
348.1824; found 348.1819. RP-C18-HPLC: 97.492%, tR = 18.891 min.
7
8
9
10-Methyl-5-(2-dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one
(5eb). Following the general procedure, 4e (1.48 g, 5 mmol) and 3-dimethylamino-
-propylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a
pale yellow solid (1.30 g, 72% yield), mp 191~193 °C; 1H NMR (500 MHz, CD3OD)
δ 8.77~8.65 (m, 1H), 7.97~7.91 (m, 2H), 7.80~7.75 (m, 2H), 7.53 (d, J = 1.7 Hz, 2H),
3.68 (t, J = 6.4 Hz, 2H), 3.00 (t, J = 6.5 Hz, 2H), 2.74 (s, 6H), 2.43 (s, 3H), 2.17~2.06
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
(m, 2H). C NMR (125 MHz, CD3OD) δ 160.2, 152.0, 145.6, 143.1, 137.6, 136.8,
133.8, 133.6, 130.1, 128.3, 127.6, 126.7, 123.6, 122.5, 120.2, 56.2, 44.2, 39.7, 27.2,
21.5. HRMS (ESI) m/z: calcd for C21H24N5O [M+H]+ 362.1981; found 362.1960.
RP-C18-HPLC: 97.174%, tR = 19.311 min.
10-Methyl-5-(3-morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin-8-
-one (5ec). Following the general procedure, 4e (1.48 g, 5 mmol) and 3-morpholino-
-propan-1-amine (1.44 g, 10 mmol) were used, and the desired product was obtained
1
as a pale yellow solid (1.70 g, 84% yield), mp 189~191 °C; H NMR (500 MHz,
CDCl3) δ 8.99~8.92 (m, 1H), 8.22 (d, J = 0.6 Hz, 1H), 7.98~7.91 (m, 1H), 7.81~7.76
(m, 2H), 7.71 (d, J = 8.4 Hz, 1H), 7.58 (dd, J = 8.4, 2.1 Hz, 1H), 7.48 (s, 1H), 3.89 (t,
J = 4.6 Hz, 4H), 3.80 (d, J = 4.5 Hz, 2H), 2.81~2.75 (m, 2H), 2.72 (s, 4H), 2.52 (s,
13
3H), 2.06~1.97 (m, 2H). C NMR (125 MHz, CDCl3) δ 158.6, 148.9, 144.4, 142.5,
135.9, 135.4, 132.1, 131.9, 129.5, 127.1, 127.0, 126.5, 121.9, 121.7, 120.1, 66.4, 58.6,
53.6, 42.2, 23.1, 21.4. HRMS (ESI) m/z: calcd for C23H26N5O2 [M+H]+ 404.2086;
28
ACS Paragon Plus Environment

Page 29 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
found 404.2077. RP-C18-HPLC: 97.628%, tR = 19.219 min.
6
7
8
10-Methyl-5-(morpholin-4-yl)phthalazino[1,2-b]quinazolin-8-one
(5ed).
9
Following the general procedure, 4e (1.48 g, 5 mmol) and morpholine (0.87 g, 10
mmol) were used, and the desired product was obtained as a pale yellow solid (1.51 g,
87% yield), mp 217~218 °C; 1H NMR (500 MHz, CDCl3) δ 8.98 (dd, J = 7.9, 1.1 Hz,
1H), 8.26 (s, 1H), 7.94 (dd, J = 7.8, 1.1 Hz, 1H), 7.87~ 7.76 (m, 2H), 7.75 (d, J = 8.3
Hz, 1H), 7.63 (dd, J = 8.4, 2.0 Hz, 1H, 3.99 (t, J = 6.0 Hz, 4H), 3.51 (t, J = 6.5 Hz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
4H), 2.54 (s, 3H). C NMR (125 MHz, CDCl3) δ 167.8, 158.8, 154.5, 144.7, 143.0,
136.5, 136.1, 132.4, 131.0, 129.0, 127.3, 126.9, 125.2, 122.7, 120.4, 66.8, 65.7, 51.5.
HRMS (ESI) m/z: calcd for C20H19N4O2 [M+H]+ 347.1508; found 347.1503.
RP-C18-HPLC: 99.167%, tR = 25.378 min.
10-Methyl-5-(4-methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5ee).
Following the general procedure, 4e (1.48 g, 5 mmol) and 1-methylpiperazine (1.00 g,
10 mmol) were used, and the desired product was obtained as a pale yellow solid
(1.51 g, 84% yield), mp 217~219 °C; 1H NMR (500 MHz, CDCl3) δ 8.96 (dd, J = 7.6,
1.7 Hz, 1H), 8.25 (s, 1H), 7.92 (dd, J = 7.6, 1.1 Hz, 1H), 7.83~ 7.76 (m, 2H), 7.74 (d,
J = 8.3 Hz, 1H), 7.62 (dd, J = 8.4, 2.1 Hz, 1H), 3.56 (s, 4H), 2.74 (s, 4H), 2.54 (s, 3H),
2.43 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 158.8, 154.5, 144.7, 143.0, 136.4, 136.0,
132.3, 132.1, 131.0, 127.3, 127.1, 126.9, 125.3, 122.8, 120.4, 54.9, 50.7, 46.2, 21.6.
HRMS (ESI) m/z: calcd for C21H22N5O [M+H]+ 360.1824; found 360.1817.
RP-C18-HPLC: 97.873%, tR = 18.897 min.
10-Bromo-5-((2-(diethylamino)ethyl)amino)-8H-phthalazino[1,2-b]quinazolin-8-
29
ACS Paragon Plus Environment